PB2235: COMPLETE DELETION OF THE CACCC BOX OF THE HBB GENE PROMOTER: A NEW MECHANISM LEADING TO A SILENT PHENOTYPE OF BETA THALASSEMIA
Authors: E. Mondesert, M. GIANSILY-BLAIZOT, J. DELAGE, F. DANTON, P. AGUILAR-MARTINEZ
Journal: HemaSphere (2022)

Background: Beta-thalassemias result from defects in the production of beta globin chains. They are inherited in an autosomal recessive manner. Heterozygotes, who are clinically asymptomatic, can typically be diagnosed by the presence of a microcytic hypochromic anemia associated with elevated HbA2 (above 3.5%), HbF being inconsistently increased. The spectrum of molecular defects is broad and heterogeneous. Exonic, intronic and flanking regions of the HBB gene, as well as distant regulatory areas on the beta globin locus control region (LCR) may be affected. Aims: We describe for the first time a complete deletion of the CACCC box of the beta globin gene promoter, which lead to a silent beta thalassemia phenotype and compared this presentation with Single Nucleotide Variants at this box and with mouse models. Methods: The proband was referred to our specialized consultation to explore an isolated increase in HbA2 (3.8 and 4.1% on 2 different controls) found at hemoglobin studies, in the context of a medically assisted procreation (MAP). The blood count was normal (Hb 156 g/L, MCV 86 fL). The patient was not taking any medication. Ferritin, thyroid hormone, vitamin B12 and B9 levels were normal. After informed written consent, molecular analysis of the beta and alpha globin gene clusters was performed, including HBB, HBA1 and HBA2 gene sequencing by the Sanger method and searching for large rearrangements (SALSA MLPA Probemix P140 HBA, and SALSA MLPA Probemix P102 HBB, MRC Holland). Family members were also examined. Results: DNA sequencing revealed an undescribed 15-base pair deletion in the HBB gene promoter: [HBB:c.-145_-131del] in the heterozygous state. This deletion removes the entire proximal CACCC motif, a binding site for the erythroid Kruppel-like factor transcription factor (EKLF/KLF1). We ruled out the coexistence of alpha-thalassemia, which would have reduced the imbalance of the alpha and beta chains, by checking the absence of pathogenic sequence variations within the alpha globin genes, as well as of large rearrangement of the alpha globin gene cluster. Family screening showed that the variant was inherited from the proband’s mother who had increased Hb A2 and normal CBC, while the father had a strictly normal profile. Genetic counseling was offered to the proband and affected family members. Summary/Conclusion: The HBB gene promoter has two CACCC motifs: a distal one located at positions c.-155_-151, where a single sequence variation, associated with silent beta-thalassemia, has been described and a proximal one, at positions -140_-136, where 7 beta + thalassemia point mutations have been reported. EKLF, which normally binds the deleted region, is involved in the regulation of several genes through its C2H2 zinc finger domain recognizing canonical CCNCNCCC sites. It plays a critical role in erythroid differentiation, and EKLF -/- mice display a beta-thalassemia phenotype and die early from anemia. However, the in vitro deletion of the CACCC box of the HBB gene shows that EKLF may also be involved in the regulation of the beta globin gene independently of the proximal CACCC motif, a hypothesis that suggests the role of other upstream CACCC elements on the LCR. The absence of anemia and/or microcytosis in the proband also contrasts with the complete loss of the proximal CACCC motif. The complete deletion of the proximal CACCC motif, a natural variant described here for the first time in the heterozygous state, in a patient with a silent beta thalassemia phenotype, confirms that this motif is not essential for the action of the erythroid transcription factor EKLF/KLF1.